An atypical NLR protein modulates the NRC immune receptor network in Nicotiana benthamiana

The NRC immune receptor network has evolved in asterid plants from a pair of linked genes into a genetically dispersed and phylogenetically structured network of sensor and helper NLR (nucleotide-binding domain and leucine-rich repeat-containing) proteins. In some species, such as the model plant Nicotiana benthamiana and other Solanaceae, the NRC (NLR-REQUIRED FOR CELL DEATH) network forms up to half of the NLRome, and NRCs are scattered throughout the genome in gene clusters of varying complexities. Here, we describe NRCX, an atypical member of the NRC family that lacks canonical features of these NLR helper proteins, such as a functional N-terminal MADA motif and the capacity to trigger autoimmunity. In contrast to other NRCs, systemic gene silencing of NRCX in N. benthamiana markedly impairs plant growth resulting in a dwarf phenotype. Remarkably, dwarfism of NRCX silenced plants is partially dependent on NRCX paralogs NRC2 and NRC3, but not NRC4. Despite its negative impact on plant growth when silenced systemically, spot gene silencing of NRCX in mature N. benthamiana leaves doesn’t result in visible cell death phenotypes. However, alteration of NRCX expression modulates the hypersensitive response mediated by NRC2 and NRC3 in a manner consistent with a negative role for NRCX in the NRC network. We conclude that NRCX is an atypical member of the NRC network that has evolved to contribute to the homeostasis of this genetically unlinked NLR network.


Introduction
Plants are invaded by a multitude of pathogens and pests, some of which threaten food security in recurrent cycles of destructive epidemics. Yet, most plants are resistant to most parasites through their highly effective immune system. Plant defense consists of an expanded and diverse repertoire of immune receptors: cell-surface pattern recognition receptors (PRRs) and intracellular nucleotide-binding domain and leucine-rich repeat-containing (NLRs) proteins [1]. Pathogen-associated molecular patterns (PAMPs) are recognized in the extracellular space by PRRs, resulting in pattern-triggered immunity (PTI) [2,3]. NLRs perceive pathogen secreted proteins known as effectors and induce robust immune responses that generally include hypersensitive cell death [4][5][6]. NLR-mediated immunity (also known as effector-triggered immunity) can be effective in restricting pathogen infection at invasion sites, and NLRs have also been recently shown to be involved in PRR-mediated signalling [7][8][9][10]. However, NLR-mediated immunity comes at a cost for plants. NLR mis-regulation and inappropriate activation can lead to deleterious physiological phenotypes, resulting in growth suppression and autoimmunity [11], and the evolution of the plant immune system is constrained by fitness trade-offs between growth and immunity [12,13]. However, our knowledge of the mechanisms by which diverse plant NLRs are regulated is still somewhat limited. Understanding how plants maintain NLR network homeostasis should help guide breeding disease resistant crops with limited fitness penalties.
NLRs occur across all kingdoms of life and generally function in innate immunity through "non-self" perception of invading pathogens [4,14,15]. Plant NLRs share a multidomain architecture typically consisting of a central NB-ARC (nucleotide-binding domain shared with APAF-1, various R-proteins and CED-4) and a C-terminal leucine-rich repeat (LRR) domain [16]. Plant NLRs can be sorted into sub-classes based on NB-ARC phylogenetic clustering and the type of N-terminal domain they carry [16,17]. The largest class of NLRs are the CC-NLRs with the Rx-type coiled-coil (CC) domain preceding the NB-ARC domain [16,18,19]. A prototypical ancient CC-NLR is the HOPZ-ACTIVATED RESISTANCE1 (ZAR1), which has remained relatively conserved throughout evolution over tens of millions of years [20,21]. However, the majority of CC-NLRs have massively expanded throughout their evolution, acquiring new activities through sequence diversification and integration of extraneous domains, as well as losing particular molecular features following sub-functionalization [22][23][24].
One class of MADA-CC-NLRs are the NRCs (NLR-REQUIRED FOR CELL DEATH), that are central nodes in a large NLR immune network of asterid plants [53]. NRCs function as helper NLRs (NRC-H), required for a large number of sensor NLRs (NRC-S) to induce the hypersensitive response and immunity [53]. These NRC-S are encoded by classical disease resistance genes that detect pathogens as diverse as viruses, bacteria, oomycetes, nematodes and insects. NRC-H and NRC-S form phylogenetic sister clades within a wider NRC superclade that makes up to half of the NLRome in the Solanaceae. This NRC superclade emerged early in asterid evolution about 100 million years ago from an ancestral pair of genetically linked NLRs [53]. However, in sharp contrast to paired NLR pairs, functionally connected NRC-H and NRC-S genes are not always clustered and can be scattered throughout the plant genomes [53]. Our current model is that the NRCs and their sensor evolved from bi-functional NLRs that have sub-functionalized and specialized throughout evolution [24,26,53]. In support of this model, the MADA sequence has degenerated in NRC-S in contrast to the NRC-H, which carry functional N-terminal α1 helices [24].
Plant-pathogen coevolution has driven NLRs to form immune receptor networks [25,27]. The emerging paradigm in the field of plant immunity is that helper NLRs, NRCs, as well as the RESISTANCE TO POWDERY MILDEW 8 (RPW8)-type CC-NLRs (CC R -NLRs) ADR1 and N REQUIREMENT GENE 1 (NRG1), form receptor networks with multiple sensor NLRs [54]. These genetically dispersed NLR networks are likely to cause a heightened risk of autoimmunity during plant growth and development. Yet, our knowledge of the regulatory mechanisms that attenuate such deleterious effects of NLR networks, especially Solanaceae NRC networks, are limited. Here, we describe NRCX, an atypical NLR protein that belongs to the NRC-H phylogenetic clade. Gene silencing of NRCX markedly impairs plant growth, presumably because of mis-activation of its helper NLR paralogs NRC2 and NRC3. We propose that NRCX maintains NRC network homeostasis by modulating the activities of key helper NLR nodes during plant growth.

Systemic silencing of NRCX impairs Nicotiana benthamiana growth
While performing virus-induced gene silencing (VIGS) of NRC family genes in the model plant Nicotiana benthamiana (S1 Fig), we found that silencing of NLR NbS00030243g0001.1 One leaf per plant was harvested from the same position (the 5th leaf from cotyledons) and was used for measuring the leaf diameter. Data was obtained from 18 different VIGS plants in three independent experiments. Statistical differences among the samples were analysed with Tukey's HSD test (p<0.01). (C) The number of up-regulated genes (log 2 (TRV:NRCX/TRV:GUS) ≧ 1 and P-value ≦ 0.05) and down-regulated genes (log 2 (TRV:NRCX/TRV:GUS) ≦ -1 and P-value ≦ 0.05) in TRV:NRCX leaf tissue compared to TRV:GUS control. (D) Enriched GO terms in up-regulated and down-regulated genes identified in C.
https://doi.org/10.1371/journal.pgen.1010500.g001 (referred to from here on as NRCX) causes a severe dwarf phenotype (Fig 1A and 1B [53,55]. Yet, none of the NRC-H and NRC-S silenced N. benthamiana plants showed quantitative growth defects (Fig 1A and 1B). These results suggest that NRCX is unique among NLR genes described to date as a "lethal-phenotype" gene according to the definition of Lloyd et al. [47].
To determine whether constitutive defense activation occurs in the dwarf plants of TRV: NRCX, total RNA was extracted from leaf tissue of four-week-old TRV:NRCX and TRV:GUS plants and were subjected to RNA-seq analysis. In total, 3162 genes were detected as differentially expressed genes (DEGs, TRV:NRCX vs. TRV:GUS), in which 1445 and 1717 genes were up-regulated and down-regulated, respectively (Fig 1C, S1 and S2 Files). The DEGs do not include NRC-H genes such as NRC2, NRC3 and NRC4 (S1 and S2 Files). Gene Ontology (GO) analysis showed significant enrichment of GO terms 'response to wounding' and 'response to biotic stimulus' in up-regulated DEGs, while 'cell redox homeostasis' is enriched in down-regulated DEGs (Fig 1D). DEGs having the GO terms 'response to wounding' and 'response to biotic stimulus' include genes encoding Proteinase inhibitor I and pathogenesis related proteins (S3 File). These results suggest that constitutive defense response is induced by NRCX systemic silencing, thereby resulting in the dwarf phenotype in N. benthamiana plants.

NRCX is a CC-NLR in the NRC helper phylogenetic clade
We investigated the precise phylogenetic position of NRCX in the NRC superclade. First, we built a phylogenetic tree with 431 CC-NLRs, including the CC-NLRome from four representative plant species (Arabidopsis, sugar beet, tomato and N. benthamiana) and 16 representative CC-NLRs (Fig 2A). Next, we extracted the NRC-H subclade NLRs, which includes NRCX, for further phylogenetic analysis (Fig 2A and 2B). In this NRC-H subclade, NRCX forms a small clade together with a tomato NLR (Solyc03g005660.3.1; named as SlNRCX) (Figs 2B and S3). The NRCX clade is more closely related to NRC1/2/3 subclade than to the NRC4 subclade ( Fig 2B).
We scanned NRCX and other NRC-H proteins for conserved sequence motifs ( Fig 2C). NRC-H members typically have the N-terminal MADA motif that is functionally conserved across many dicot and monocot CC-NLRs and is required for hypersensitive cell death and disease resistance [24]. We ran the HMMER software [56] to query NRCX with a previously reported MADA motif-Hidden Markov Model (HMM) [24]. This HMMER search predicted a MADA sequence at the N-terminus of NRCX (HMM score = 22.2) and in all other NRC-H except in four N. benthamiana NRC-H proteins that have N-terminal truncations (NbS00017924g0016.1, NbS00017801g0004.1, NbS00030989g0011.1 and NbS00020047g0002.1) (Fig 2C). In addition, NRCX carries intact P-loop and MHD motif in its NB-ARC domain like the majority of CC-NLRs ( Fig 2C). We noted that the P-loop wasn't predicted in NbS00004191g0008.1 and NbS00004611g0006.1, and the MHD motif is absent or divergent in NbS00030989g0011.1, NbS00017801g0004.1 and Solyc04g007050.3.1 (Fig 2C). Taken together, we conclude that NRCX has the typical sequence motifs of MADA-CC-NLRs, similar to the great majority of proteins in the NRC-H subclade.

Unlike other NRC helpers, mutations in the MHD motif fail to confer autoactivity to NRCX
Even though NRCX carries canonical features of MADA-CC-NLRs, we investigated whether it can execute the hypersensitive cell death similar to other NRC helper NLRs. Histidine (H) to arginine (R) or aspartic acid (D) to valine (V) substitutions within the MHD motif confer autoactivity to the NRC helpers NRC2, NRC3 and NRC4 [57]. To test if NRCX has cell death induing activity like other helper NRCs, we performed site-directed mutagenesis of the MHD motif predicted in the NB-ARC domain of NRCX. We first introduced the H474R and D475V mutations in NRCX (referred to as NRCX HR and NRCX DV , respectively) ( Fig 3A). Both NRCX HR and NRCX DV did not induce macroscopic cell death when expressed in N. benthamiana leaves using agroinfiltration, in contrast to an NRC4 autoactive MHD motif mutant (NRC4 DV ) ( Fig 3B). To further challenge this finding, we randomly mutated NRCX H474 and D475 residues in the MHD motif and used agroinfiltration to express them in N.

The N-terminal MADA motif of NRCX is not functional and doesn't mediate hypersensitive cell death
To further investigate why NRCX cannot execute the cell death activity, we determined whether or not the MADA motif of NRCX is functional using the motif swap strategy we previously developed [24]. To this end, we swapped the first 17 amino acids of the autoactive NRC4 DV with the equivalent region of NRCX, resulting in a MADA NRCX -NRC4 DV chimeric protein (Fig 4A and 4B). The N-terminal sequence of NRC4 can be functionally replaced with matching sequences of other MADA-CC-NLRs, including NRC2 from N. benthamiana, ZAR1 from Arabidopsis and even the monocot MADA-CC-NLRs MLA10 and Pik-2 [24]. Intriguingly, MADA NRCX -NRC4 DV did not induce any visible cell death when expressed by agroinfiltration in N. benthamiana leaves unlike the positive controls MADA NRC2 -NRC4 DV and MADA ZAR1 -NRC4 DV (Fig 4A-4C). MADA NRCX -NRC4 DV chimeric protein accumulated to similar levels as MADA ZAR1 -NRC4 DV in N. benthamiana leaves, indicating that the lack of activity was probably not due to protein destabilization ( Fig 4E). These results indicate that the MADA region of NRCX does not have the capacity to trigger hypersensitive cell death in the NRC4 protein background, and therefore despite its sequence conservation, it probably forms a non-functional N-terminal α1 helix.
We also performed the opposite motif swap to test whether functional MADA sequences confer gain of cell death activity to NRCX. To this end, we produced MADA chimera constructs in the NRCX DV

The dwarf phenotype of NRCX-silenced Nicotiana benthamiana plants is partially dependent on NRC2 and NRC3 but not NRC4
We hypothesized that NRCX has a regulatory role in the NRC network, given that it belongs to the NRC helper clade but lacks the capacity to cause hypersensitive cell death. To test this hypothesis, we silenced NRCX in three different N. benthamiana lines, nrc2/3, nrc4 and nrc2/ 3/4, that we previously described as carrying loss-of-function mutations in NRC2, NRC3 and/ or NRC4 [24,58,59] (Fig 5). Four weeks after inoculation with TRV:NRCX, we observed partial suppression of the TRV:NRCX-mediated growth defects in nrc2/3 and nrc2/3/4 plants, but not in nrc4 plants (Fig 5A-5C). We confirmed that the quantitative differences were reproducible by using at least two independent lines of each of the three mutants (Fig 5A-5C). In these experiments, we did not observe quantitative growth differences between nrc2/3 and nrc2/3/4 plants (Fig 5B and 5C).
To independently challenge these results, we performed co-silencing experiments where NRCX was knocked-down together with NRC2/3, NRC4 or NRC2/3/4. To silence multiple genes by VIGS in N. benthamiana plants, we tandemly cloned gene fragments of NRCX and other NRCs in the same TRV expression vector. Compared to TRV:NRCX plants, the dwarf phenotype was partially suppressed in TRV:NRC2/3/X and TRV:NRC2/3/4/X, but not in TRV: Each silencing construct specifically reduced mRNA levels of the target NRC gene (S8D Fig), indicating that phenotypic differences are unlikely to be due to off-target gene silencing effects. Altogether, we conclude that the TRV:NRCX-mediated dwarf phenotype is partially dependent on NRC2 and NRC3, but not NRC4.

Hairpin RNA-mediated gene silencing of NRCX in Nicotiana benthamiana leaves doesn't result in visible cell death phenotypes
To gain further insights into NRCX activities, we generated a hairpin RNA (hpRNA) silencing construct (hpRNA:NRCX) for NRCX silencing after transient expression in mature N. benthamiana leaves. First, we investigated the degree to which the hpRNA:NRCX expression causes cell death in N. benthamiana leaves, given that NLR-mediated dwarfism in plants is often linked to cell death [40]. Five days after the agroinfiltration, hpRNA-mediated gene silencing of NRCX did not result in macroscopic cell death in N. benthamiana leaves whereas

Hairpin RNA-mediated gene silencing of NRCX enhances NRC2-and NRC3-dependent cell death
Our finding that mutations in NRC2 and NRC3 are genetic suppressors of TRV:NRCX-mediated dwarfism raises the possibility that NRCX negatively modulates the activity of these helper NLRs and prompted us to investigate this hypothesis. To this end, we co-expressed hpRNA: NRCX by agroinfiltration in N. benthamiana leaves with NRC-dependent sensor NLRs (Pto/ SlPrf, Gpa2, Rpi-blb2, R1, Sw-5b and Rx) or NRC-independent NLRs (R2 and R3a) and their matching pathogen effectors [53,57]. hpRNA-mediated gene silencing NRCX enhanced the hypersensitive cell death triggered by the sensor NLRs SlPrf, Gpa2 (NRC2 and NRC3 dependent) and Sw-5b (NRC2, NRC3 and NRC4 dependent) relative to the hpRNA:GUS control, but did not affect the other sensor NLRs, including Rpi-blb2, R1, Rx, R2 and R3a (Figs 6A, 6B and S10). It should be pointed out that although NRC2, NRC3 and NRC4 redundantly contribute to effector-activated Sw-5b hypersensitive cell death [53], we recently showed that an autoactive mutant of Sw-5b (Sw-5b D857V ) signals only through NRC2 and NRC3 [57]. Therefore, we conclude that knocking-down of NRCX enhances the activities of NRC2-and NRC3-dependent NRC-S, but doesn't affect the activity of NRC-S that are only dependent on NRC4, as well as other NLR outside the NRC network.
Next, we investigated the extent to which NRCX affects the activities of autoactive mutants of the NRC-H, NRC2 H480R (NRC2 HR ), NRC3 D480V (NRC3 DV ) and NRC4 D478V (NRC4 DV ), which cause cell death even in the absence of pathogen effectors [57]. We co-expressed NRC2 HR , NRC3 DV and NRC4 DV with hpRNA:NRCX or hpRNA:GUS by agroinfiltration in N. benthamiana leaves. NRCX silencing enhanced the cell death responses caused by NRC2 HR and NRC3 DV , but not NRC4 DV , compared to the hpRNA:GUS silencing control (Fig 6C, 6D and S11). hpRNA:NRCX expression reduced mRNA levels of endogenous NRCX gene, but did not significantly alter the expression of other NRCs, indicating that the enhanced cell death phenotype was probably not due to off-target silencing (Fig 6E). Altogether, these two sets of hpRNA-mediated gene silencing experiments indicate that NRCX modulates the helper NLRs NRC2 and NRC3 nodes in the NRC network.

NRCX overexpression compromises NRC2 and NRC3, but not NRC4, autoimmune cell death
We further challenged our model by testing the extent to which NRCX overexpression can suppress the cell death caused by autoactive NRC2 and NRC3. We generated an overexpression construct of wild-type NRCX and co-expressed it by agroinfiltration with autoactive NRC2 HR , NRC3 DV and NRC4 DV mutants in N. benthamiana leaves. Five days after agroinfiltration, we observed that wild-type NRCX expression compromised autoimmune cell death triggered by NRC2 HR and NRC3 DV , but not NRC4 DV , relative to the empty vector control (Fig 7). Taken together, manipulation of NRCX expression levels in N. benthamiana leaves suggests a negative role of NRCX in NRC2 and NRC3, but not NRC4, mediated immunity.
To further test whether tomato NRCX ortholog has a similar negative role in NRC3 autoactive cell death, we cloned wild-type SlNRCX and overexpressed it with NRC3 DV mutant in N.  NRCX is differentially expressed relative to NRC2a/b following activation of pattern-triggered immunity Our finding that NRCX modulates NRC2 and NRC3 nodes prompted us to investigate the transcriptome dynamics of NRCX compared to these NRC-H in different plant tissues. To obtain transcriptome data of NRC-H clade members, we extracted total RNA from leaf, root and flower/bud of five-week-old wild-type N. benthamiana. Three replicate each from independent plants were subjected to RNA-seq and resulted in 40 million 150-bp paired-end reads per sample. Then, we calculated Transcripts Per Million (TPM) values for N. benthamiana NLR genes (S5 File). In Fig 8A, we focused on transcriptome profiles of NRC-H with a TPM > 1.0 and found that NRCX, NRC2a/b/c, NRC3 and NRC4a/b genes were expressed in all three tissues, leaf, root and flower/bud. Notably, NRCX was about 3 to 5-fold more highly expressed in roots (TPM = 25.5) compared to leaves (TPM = 7.5) and flowers/buds (TPM = 5.2) (Fig 8A). In addition to the NRC2, NRC3, NRC4 and NRCX, we also noted that NRC5a and NRC8a were expressed in the three tissues, whereas NRC7a and NRC8b were mainly expressed in N. benthamiana roots (Fig 8A). Considering that NRCX is coexpressed with NRC2 and NRC3 in leaf, root and flower/bud, we propose that NRCX maintains NRC2/3 subnetwork homeostasis during these developmental stages.
Next, we analysed transcriptome data of six-week-old wild-type N. benthamiana leaves upon inoculation with bacteria Pseudomonas fluorescens 55 [60]. P. fluorescens 55 inoculation is considered to trigger PTI in N. benthamiana. We, therefore, explored the degree to which NRC-H genes are up-or down-regulated upon PTI activation. In total, 61 NLR genes were upregulated, while 14 NLR genes were down-regulated in bacterial vs. mock treatment (Fig 8B). Interestingly, the expression of NRC2a/b genes was up-regulated in response to P. fluorescens 55 inoculation whereas there were no particular differential expression changes with NRCX ( Fig 8B, S1 Table). Other NRC-H genes, such as NRC2c, NRC3 and NRC4a/b, were also unchanged whereas the expression level of NRC5a/b increased following bacterial treatment ( Fig 8B). We conclude that following activation of immunity in response to bacterial inoculation, NRC2a/b become more highly expressed than their paralog and modulator NRCX, thereby altering the balance of gene expression between NRC2a/b and NRCX.

Discussion
Co-operating plant NLRs are currently categorized into "sensor NLR" for effector recognition or "helper NLR" for immune signalling [54]. These functionally specialized NLR sensors and helpers function in pairs or networks across many species of flowering plants. In this study, we found that NRCX, a recently diverged paralog of the NRC class of helper NLRs, contributes to sustain proper plant growth and is a modulator of the genetically dispersed NRC2/3 subnetwork. We propose that NRCX has evolved to maintain the homeostasis of at least a section of the NRC network (Fig 9). NRCX is also atypical as far as NLR helpers and NRC proteins go, lacking a functional N-terminal MADA motif and the capacity to trigger hypersensitive cell death. At the moment, we cannot categorize NRCX as either a sensor or helper NLR, and it is best described as an NLR modulator.
NLRs are often implicated in spontaneous autoimmune phenotypes in plants and humans [11,61]. In plants, autoimmunity is often observed at the F1 and later generations when genetically distinct plant accessions are crossed [11][12][13]. One well-studied case of hybrid autoimmunity is induced by a hetero-complex of NLRs from genetically unlinked NLR gene loci between DANGEROUS MIX 1 (DM1) from Arabidopsis accession Uk-3 and DM2 from Uk-1 [62][63][64]. Therefore, hybrid incompatibility can be due to inappropriate activation of mismatched NLRs. Amino acid insertions or substitutions in NLR genes can also exhibit autoimmune phenotypes in Arabidopsis, such as in the NLR alleles ssi4 (G422R), snc1 (E552K), slh1 (single leucine insertion to RRS1 WRKY domain), chs1 (A10T), chs2 (S389F) and chs3-2D (C1340Y) [31][32][33][34]36,37]. In chs3-1, a truncation of the C-terminal LIM-containing domain causes autoimmunity [35]. However, to date, T-DNA insertion or deletion mutants including chs3-2, where expression of full-length NLR modules is suppressed, do not show autoimmune phenotypes [32,35,43,65]. Therefore, autoimmune NLR mutants are typically considered gain-of-function mutants. A striking finding in this study is that systemic NRCX silencing resulted in severe dwarfism (Fig 1A and 1B). The dwarf TRV:NRCX plants showed constitutive activation of defense-related genes (Fig 1C and 1D). To our knowledge, this finding is a unique example where VIGS of an NLR gene causes a "lethal" plant phenotype, following the definition by Lloyd et al. [47]. Therefore, NRCX is exceptional given that NLR genes tend to act just the opposite way, by causing fitness penalties.
Mutations in NRC2 and NRC3 partially suppress the dwarf phenotype of NRCX-silenced plants and can be viewed as genetic suppressors of NRCX (Fig 5). These findings inspired us to draw a model which expands our understanding of the NRC network to include NRCX as a modulator of the NRC2 and NRC3 nodes (Fig 9). This network even connects NLRs to cellsurface immune receptors, given that Kourelis et al. [10] recently showed that NRC3 is required for the hypersensitive cell death triggered by the receptor protein Cf-4. We propose that NRCX contributes to the homeostasis of the NRC2/3 subnetwork, which are hubs in an immune network composed of both intracellular NRC-S and the cell-surface receptors. When the expression level of NRCX is reduced, NRC2 and NRC3 cause autoimmunity, possibly through inadvertent activation (Fig 9). However, hpRNA-mediated gene silencing of NRCX in N. benthamiana leaves did not cause autoimmune cell death, although it enhanced the hypersensitive cell death elicited by effector recognition (Figs 6 and S9). This indicates that NRCXmediated dwarfism may be dependent on existence of environmental microbes or viruses and may occur in particular tissues and/or during certain developmental stages. Considering that nrc2/nrc3 knockout plants do not fully recover from dwarfism (Fig 5), it is possible that NRCX silencing leads to a permanent "trigger-happy" status of NRC2/3 and other NLR(s) that ultimately perturbs plant growth.
NRCX clusters within the NRC-H subclade, which is populated with proteins with a MADA-CC-NLR architecture (Fig 2). However, the N-terminal MADA motif of NRCX was not functional when swapped into NRC4 (Fig 4), compared to other similar swaps we previously tested [10,24,66]. We conclude that the MADA motif of NRCX has degenerated from the canonical sequence present in the typical NRC helper NLRs and lost the capacity to execute the hypersensitive cell death response. In fact, a polymorphism in the NRCX MADA motif region, Thr-4, is unique to this protein among NRC-H (Fig 2). Moreover, given that functional MADA sequences did not confer cell death activity to NRCX (S7 Fig), NRCX may have become inactive to trigger cell death response by other mutation(s). Therefore, loss of cell death executor activity in NRCX might represent another path in the evolution of networked NLRs besides sensor and helper NLRs, as in the NLR functional specialization "use-it-or-loseit" model described by Adachi et al. [24] and Kamoun [67]. We propose that NRCX has also functionally specialized into an atypical NLR protein that operates as an NLR modulator.
N. benthamiana and tomato NRCX overexpression compromised but did not fully suppress NRC3 autoactive cell death (Figs 7 and S12). Its suppressor activity contrasts with that of the SPRYSEC15 effector from the cyst nematode Globodera rostochiensis and AVRcap1b from the oomycete Phytophthora infestans, which strongly suppress the cell death inducing activity of autoimmune NRC2 and NRC3 [57]. In the future, it will be interesting to determine the mechanism by which NRCX modulates the activities of NRC-H proteins, and how that process compares with pathogen suppression of the NRC network.
Our work on NRCX adds to a growing body of knowledge about NLRs that modulate the activities of other NLRs. Classic examples include genetically linked NLR pairs, such as the Pia RGA5/RGA4 pair, in which the sensor RGA5 suppresses the RGA4 autoimmune cell death observed in N. benthamiana [68]. In this and other one-to-one paired NLRs, the sensor NLR carries a regulatory role of its genetically linked helper mate that is released by the matching pathogen effector. In contrast, NRCX modulates a genetically dispersed NLR network composed of a large number of sensor NLRs and two helper NLRs, NRC2 and NRC3. Recently, Wu et al. [69] showed that overexpression of NRG1C, antagonizes autoimmunity by its paralog NRG1A, chs3-2D and snc1, without affecting chs1, chs2 autoimmunity and RPS2-and RPS4-mediated immunity. NRG1 is a member of CC R -NLR helper subfamily that triggers cell death via its N-terminal CC R domain [70]. Intriguingly, unlike NRCX, NRG1C has a truncated NLR architecture, lacking the whole N-terminal CC R domain and is therefore unlikely to execute the hypersensitive cell death [69]. The emerging picture is that NRCX and NRG1C are an atypical subclass of helper NLRs, we term modulator NLRs, that lost their cell death executor activity through regressive evolution and evolved to modulate the activities of multiple NLR helper proteins. These examples form another case of functional specialization during the transitions associated with NLR evolution [67]. We need a better appreciation of the diversity of structures and functions that come with the protein we classify as NLR immune receptors and integrate the different ways NLRs contribute to immunity [26].
Plant NLRs are tightly regulated at the transcript level because increased expression of NLRs can result in autoimmune phenotypes and fitness costs [71][72][73][74]. However, activation of plant defense is associated with massive up-regulation of NLR genes. For instance, in Arabidopsis, dozens of NLR genes are up-regulated in response to pathogen-related treatments, such as the bacterial PAMP flg22 [75,76]. The current model is that NLR expression level is maintained at a low basal level but is amplified in the cells upon activation of pathogeninduced immunity. In our analyses, we found that 61 NLR genes, including NRC2a/b, are upregulated in N. benthamiana leaves following P. fluorescens inoculation, while NRCX expression levels remain unchanged (Fig 8B). This marked shift in the balance between NRC2a/b to NRCX expression levels may potentiate and amplify the activity of the NRC network resulting in more robust immune responses (Fig 9).
The NRC superclade forms a large and complex NLR immune receptor network in asterid plants that connects to cell surface receptors [10,53]. Here we show that NRCX modulates the hub NLR proteins NRC2 and NRC3, but doesn't affect their paralog NRC4. Further studies are required to determine the molecular mechanisms underpinning NRCX antagonism of NRC2and NRC3-mediated immune response and what other mechanisms modulate other sections of the network, such as the NRC4 subnetwork. Our findings also highlight the potential fitness costs associated with expanded NLR networks as the risk of inadvertent activation increases with the network complexity. Further understanding of how NLR network homeostasis is maintained will provide insights for future breeding of vigorous and disease resistant crops.
To generate MHD mutants of NRCX, the histidine (H) and aspartic acid (D) residues in the MHD motif were substituted by overlap extension PCR using Phusion High-Fidelity DNA Polymerase (Thermo Fisher). The pICSL01005::NRCX without its stop codon was used as a template. The mutagenesis primers are listed in S2 Table. The mutated NRCX was verified by DNA sequencing of the obtained plasmids.
To generate the hpRNA-mediated gene silencing construct, the silencing fragment (shown in S2 Fig) was amplified from NRCX cDNA by Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) using primers listed in S2 Table. The purified amplicon was cloned into the pRNAi-GG vector [79].
Information of other constructs used for the cell death assays were described in S3 Table. Virus-induced gene silencing (VIGS) VIGS was performed in N. benthamiana as previously described [80]. Suspensions of Agrobacterium Gv3101 strains carrying TRV RNA1 and TRV RNA2 were mixed in a 1:1 ratio in infiltration buffer (

Bioinformatic and phylogenetic analyses
Based on the NLR annotation and phylogenetic tree previously described in Harant [16] to protein databases annotated in each reference genome (S7 File). Amino acid sequences of the annotated NLR genes from the twelve plant species are listed in S4 File. Amino acid sequences of the NLR datasets were aligned using MAFFT v.7 [82]. The gaps in the alignments were deleted manually and the NB-ARC domain sequences were used for generating phylogenetic tree (S8 and S9 Files). The maximum likelihood tree based on the JTT model was made in RAxML version 8.2.12 [83] and bootstrap values based on 100 iterations were shown in S10 and S11 Files. NRC-helper proteins were subjected to motif searches using MEME (Multiple EM for Motif Elicitation) v. 5.0.5 [84] with parameters 'zero or one occurrence per sequence, top twenty motifs', to detect consensus motifs conserved in > 80% of input sequences.

Transient gene expression and cell death assays
Transient gene expression in N. benthamiana was performed by agroinfiltration according to methods described by Bos et al. [85]. Briefly, four-week-old N. benthamiana plants were infiltrated with Agrobacterium Gv3101 strains carrying the binary expression plasmids. The Agrobacterium suspensions were prepared in infiltration buffer (10 mM MES, 10 mM MgCl 2 , and 150 μM acetosyringone, pH5.6). To overexpress NRC MADA chimeras and MHD mutants, the concentration of each suspension was adjusted to OD 600 = 0.5. To perform hpRNA-mediated gene silencing experiments in N. benthamiana leaves, we infiltrated Agrobacterium strains carrying hpRNA constructs (OD 600 = 0.5), together with different proteins described in S3 Table. Macroscopic cell death phenotypes were scored according to the scale of Segretin et al. [86] modified to range from 0 (no visible necrosis) to 7 (fully confluent necrosis).
To stain dead cells by trypan blue, N. benthamiana leaves were transferred to a trypan blue solution (10 mL of lactic acid, 10 mL of glycerol, 10 g of phenol, 10 mL of water, and 10 mg of trypan blue) diluted in ethanol 1:1 and were incubated at 65˚C using a water bath for 1 hour. The leaves were then destained for 48 hours in a chloral hydrate solution (100 g of chloral hydrate, 5 mL of glycerol, and 30 mL of water).

RNA extraction and semi-quantitative RT-PCR
Total RNA was extracted using RNeasy Mini Kit (Qiagen). 500 ng RNA of each sample was subjected to reverse transcription using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific). Semi-quantitative reverse transcription PCR (RT-PCR) was performed using DreamTaq (Thermo Fisher Scientific) with 25 to 30 amplification cycles followed by electrophoresis with 1.8% (w/v) agarose gel stained with Ethidium bromide. Primers used for RT-PCR are listed in S2 Table. RNA-seq analysis Total RNAs of leaf tissue samples were extracted from four-week-old TRV:GUS and TRV: NRCX benthamiana plants using RNeasy Mini Kit (Qiagen) or using TRI Reagent (Sigma-Aldrich) as directed in the protocol. Total RNAs of leaf, root and flower/bud tissue samples were extracted from five-week-old N. benthamiana plants using RNeasy Mini Kit (Qiagen). Three replicate each of the samples was sent for Illumina NovaSeq 6000 (40 M paired-end reads per sample, Novogene). Obtained RNA-seq reads were filtered and trimmed using FaQCs [87]. The quality-trimmed reads were mapped to the reference N. benthamiana genome (Sol Genomics Network, v0.4.4) using HISAT2 [88]. The number of read alignments in the gene regions were counted using featureCounts [89] and read counts were transformed into a Transcripts Per Million (TPM) value. Differentially expressed genes between TRV:GUS and TRV:NRCX plants were determined by edgeR through a threshold of log 2 FC and false discovery rate (|log 2 FC| > 1 and FDR < 0.05) [90]. For GO analysis, we used GO annotation list (S12 File) extracted from Nicotiana benthamiana v0.4.4 database (Solanaceae Genomics Network) and ran g:GOSt tool in g:Profiler [91] with parameters 'ordered query, only annotated genes, g:SCS threshold, threshold < 0.05' to detect enriched GO terms in differentially expressed gene datasets. Public RNA-seq reads from six-week-old N. bethamiana leaves with or without Pseudomonas fluorescens 55 inoculation [60], were also analysed as described above (Accession Numbers: SRP118889).
RNA-seq raw reads used for transcriptomic analyses in this study have been deposited under the BioProject accessions: TRV:GUS_leaf and TRV:NRCX_leaf (PRJEB55392), and fiveweek old wild-type N. benthamiana_leaf, root and flower/bud (PRJEB55516). NRC sequences used in this study can be found in the GenBank/EMBL and Solanaceae Genomics Network